Methods of Using Ryanodine Antagonists in Treating Neural Injury

ABSTRACT

The present invention provides a method of providing neuroprotection to a mammal comprising administering to said mammal suffering from or at risk of suffering a noxious action on its nerve cells an effective amount of a ryanodine antagonist, e.g. dantrolene, to inhibit or prevent nerve cell injury or death.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. Ser. No. 10/189,676, filed Jul. 3, 2002,which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to neurology and ophthalmology, and morespecifically to protection of neural tissues from injuries caused byabnormal elevation of intracellular free calcium through calcium releasefrom intracellular stores under disease conditions, including stroke,acute brain trauma, Alzheimer's disease, Parkinson's disease, glaucoma,diabetic retinopathy, and age-related macular degeneration.

BACKGROUND OF THE INVENTION

There is compelling evidence that abnormally elevated intracellular freecalcium is one of the early events in the chain of reactions leading toneuronal damage under pathological conditions that range from acuteneural injuries, such as stroke, to more chronic indications, such asAlzheimer's disease. High intracellular free calcium can causemitochondrial injury and activate various types of enzymes, such asproteases, nitric oxide synthases and endonucleases. Thesecalcium-induced/activated cellular responses are believed to mediatecytotoxicity that eventually leads to neuronal death.

There are two major mechanisms that can cause elevation of intracellularfree calcium: 1) calcium influx from extracellular space through calciumand non-selective cation channels on the cell membrane, and 2) calciumrelease from intracellular stores, such as endoplasmic reticulum andmitochondria, through specialized receptor-channel complex, such asryanodine receptor channels. These two mechanisms often interact. Forexample, calcium entered the cell through ion channels on the cellmembrane can trigger more calcium release from intracellular stores.This calcium-induced calcium release (CICR) has been demonstrated tocontribute to neuronal damage under pathological conditions.

Glutamate is the major excitatory neurotransmitter in the brain,including the retina. Its biological action is mediated by a variety ofglutamate receptors, including the NMDA receptor that is an ionotropicreceptor coupled with a non-selective cation channel that has highcalcium permeability. Under pathological conditions, glutamate becomes aneurotoxin that causes neuronal damage in both acute neural injuries,such as stroke, to more chronic indications, such as Alzheimer'sdisease. This glutamate excitotoxicity is mediated, to a large extent,by the NMDA receptor because of its high calcium permeability. Overstimulation of the NMDA receptor resulting from either excessive releaseor reduced reuptake of glutamate causes intracellular calcium overloadthat can eventually lead to neuronal death. Calcium entering the neuronthrough NMDA channels can stimulate more calcium release fromintracellular stores via specialized ligand-activated channels, such asryanodine channels. This calcium-induced calcium release amplifiescellular response triggered by NMDA receptor activation and has beenshown to contribute to excitotoxicity under pathological conditions.

Glaucoma is a neurodegenerative retinal disease characterized byprogressive death of retinal ganglion cells (RGCs, the output neuron ofthe retina), which leads to progressive vision loss and eventually tocomplete blindness. Glaucoma can be classified into two majorcategories: hypertensive and normotensive. The underlying causes forglaucoma are still not well understood. The initial insults for the twotypes of glaucoma are likely different. High intraocular pressure isbelieved to be a major risk factor for the hypertensive glaucoma whereasthe vascular abnormality is though to play a significant role ininitiation and progression of the normotensive glaucoma. Despite thedifference in initial insults, progressive death of RGCs appears to be acommon feature shared by both types of glaucoma.

There is increasing evidence that glutamate-induced excitotoxicity playsa significant role in the pathology of glaucoma. It has beendemonstrated that glutamate concentration in vitreous humor from theglaucoma patients is significantly higher than that of normal subjectsand the vitreal glutamate concentration increases with the years withglaucoma. It has also been shown that the NMDA receptor antagonist,memantine, ameliorates RGC loss in glaucomatous monkeys, suggesting thatthe NMDA receptor mediates, at least in part, glutamate-induced damageto RGCs in glaucoma.

Diabetic retinopathy is another chronic degenerative retinal diseasethat leads progressive vision loss. Recent studies provide evidence thatischemia and glutamate excitotoxicity contribute to neural injury indiabetic retinopathy. This suggests that calcium release fromintracellular stores is likely involved in the pathology of diabeticretinopathy.

Thus, it is evident that there is an unmet need for agents that haveneuroprotective effects that can stop or retard the progressive damageto CNS neurons resulting from abnormally elevated intracellular freecalcium caused by various noxious provocations.

Dantrolene, a skeletal muscle relaxant, has been found to be anantagonist of the ryanodine receptor-channel complex (See Biochemistry2001, 40, 531-542). Dantrolene blocks calcium release from ryanodinechannels when it binds to the receptor.

Dantrolene is 1-[[5-(p-Nitrophenyl)furfurylidene]amino]hydantoin.

SUMMARY OF THE INVENTION

A new method of protecting the neurons in the retina and other parts ofthe brain of a mammal from noxious provocations has been discovered. Thepresent method uses a ryanodine receptor antagonist to prevent orameliorate damage to CNS neurons caused by noxious provocations thatinduce excessive calcium release from intracellular stores via ryanodinereceptor channels. These noxious provocations, including excitotoxicity,ischemia, hypoxia, mitochondrial dysfunction, and oxidative injury, areassociated with acute and chronic neural disorders, including glaucoma,diabetic retinopathy, age-related macular degeneration (ARMD), stroke,acute brain trauma, Alzheimer's disease, Parkinson's disease, andHuntington's disease. The method comprises administering to the mammaleither systemically, topically, epidurally or by intrabulbar injectionan effective amount of one or more ryanodine receptor antagonists, suchas dantrolene (see below for details).

For protection of retinal neurons in humans suffering from glaucoma,diabetic retinopathy, and age-related macular degeneration, the activecompounds (or mixtures or salts thereof) are administered in accordancewith the present invention to the eye admixed with an ophthalmicallyacceptable carrier. Any suitable, e.g., conventional, ophthalmicallyacceptable carrier may be employed. A carrier is ophthalmicallyacceptable if it has substantially no long term or permanent detrimentaleffect on the eye to which it is administered. Examples ofophthalmically acceptable carriers include physiological saline andother aqueous media. In accordance with the invention, the activecompounds are preferably soluble in the carrier which is employed fortheir administration, so that the active compounds are administered tothe eye in the form of a solution. Alternatively, a suspension of theactive compound or compounds (or salts thereof) in a suitable carriermay also be employed.

In accordance with the invention the active compounds (or mixtures orsalts thereof) are administered in an ophthalmically acceptable carrierin sufficient concentration so as to deliver an effective amount of theactive compound or compounds to the eye. Preferably, the ophthalmic,therapeutic solutions contain one or more of the active compounds in aconcentration range of approximately 0.0001% to approximately 10%(weight by volume) and more preferably approximately 0.005% toapproximately 0.5% (weight by volume).

Any method of administering drugs directly to a mammalian eye may beemployed to administer, in accordance with the present invention, theactive compound or compounds to the eye to be treated. By the term“administering directly” is meant to exclude those general systemic drugadministration modes, e.g., injection directly into the patient's bloodvessels, oral administration and the like, which result in the compoundor compounds being systemically available. The primary effect on themammal resulting from the direct administering of the active compound orcompounds to the mammal's eye is preferably a reduction in intraocularpressure. More preferably, the active useful compound or compounds areapplied topically to the eye or are injected directly into the eye.Particularly useful results are obtained when the compound or compoundsare applied topically to the eye in an ophthalmic solution (oculardrops).

Topical ophthalmic preparations, for example ocular drops, gels orcreams, are preferred because of ease of application, ease of dosedelivery, and fewer systemic side effects, such as cardiovascularhypotension. An exemplary topical ophthalmic formulation is shown belowin Table 1. The abbreviation q.s. means a quantity sufficient to effectthe result or to make volume.

TABLE I Ingredient Amount (% W/V) Active Compound in accordance about0.0001 to about 1 with the invention, Preservative   0-0.10 Vehicle 0-40 Tonicity Adjustor  1-10 Buffer 0.01-10   PH Adjustor q.s pH4.5-7.5 Antioxidant as needed Purified Water as needed to make 100%

Various preservatives may be used in the ophthalmic preparationdescribed in Table I above. Preferred preservatives include, but are notlimited to, benzalkonium chloride, chlorobutanol, thimerosal,phenylmercuric acetate, and phenylmercuric nitrate. Likewise, variouspreferred vehicles may be used in such ophthalmic preparation. Thesevehicles include, but are not limited to, polyvinyl alcohol, povidone,hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose,hydroxyethyl cellulose, and purified water.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol, and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. Accordingly, buffersinclude but are not limited to, acetate buffers, citrate buffers,phosphate buffers, and borate buffers. Acids or bases may be used toadjust the pH in these formulations as needed.

In a similar vein, ophthalmically acceptable antioxidants include, butare not limited to, sodium metabisulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene.

The ophthalmic solution (ocular drops) may be administered to themammalian eye as often as necessary to obtain the desired concentrationintravitreally that affords neuroprotection. For acute neuroprotectiveeffect such as photoprotection in laser treatment for ARMD, theprotective agent would be administered in advance of the treatment toprovide optimal protection during the laser procedure. For chronictreatments such as in protection of the retinal ganglion cells againstdamage from the neuropathic effects of, for example, glaucoma or dryARMD, the drug would be administered as frequently as necessary tomaintain desired intravitreal concentration or range of concentrationsat all times. In other words, the ophthalmic solution (or otherformulation) which contains the ryanodine antagonist as the activeingredient, is administered to the mammalian eye as often as necessaryto maintain the beneficial neuroprotective effect of the activeingredient in the eye. Those skilled in the art will recognize that thefrequency of administration depends on the precise nature of the activeingredient and its concentration in the ophthalmic formulation. Withinthese guidelines it is contemplated that the ophthalmic formulation ofthe present invention will be administered to the mammalian eyeapproximately once or twice daily.

This new method is particularly effective when administered as aprophylactic treatment, i.e. before damage to the nerve has taken place,or before long-term progression of the disease state, such as glaucoma,diabetic retinopathy, or ARMD, has taken place. Without wishing to beheld to a particular theory regarding the role that the compounds of thepresent invention play in neuroprotection, applicants hypothesize thatthe compounds and methods described inhibit the intracellular Ca+2release. (See for example U.S. Pat. No. 5,891,911.)

Thus it is further contemplated that the compounds of the presentinvention can advantageously be used in combination with compounds thatinhibit cell death. Such cell death inhibiting compounds include NMDAantagonists especially memantine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to methods of using ryanodine receptorantagonists to protect CNS neurons, particularly the retinal neurons,from injuries caused by acute and chronic noxious provocations. Thedrawing will first be briefly described.

DRAWINGS

FIG. 1 is a bar graph showing the neuroprotective effect of dantroleneon NMDA-induced injury of retinal ganglion cells in vivo in rabbits.Intravitreal injection of NMDA caused retinal ganglion cell loss incontrol animals. Application of dantrolene ameliorated NMDA-induceddamage to ganglion cells.

As mentioned above, excessive release of calcium from intracellularstores under disease conditions is cytotoxic to neurons. NMDA receptormediated excitotoxicity is believed to be a common cause that cantrigger excessive calcium release from intracellular stores in acute andchronic disorders mentioned above. For example, NMDA receptor antagonistmemantine protects RGCs in glaucomatous monkeys, suggesting that theNMDA receptor mediates, at least in part, glutamate excitotoxicity inglaucoma.

There is strong evidence that damage to CNS neurons often has twostages: Primary and secondary degeneration. Initially, direct neuronalinsults, such as local ischemia, trauma etc., lead to degeneration ofthe affected neurons. However, the associated pathophysiological andbiochemical events occurring in the injured neurons are probablyresponsible for the subsequent progressive (secondary) degeneration ofthe neighboring neurons that are not directly affected by the primaryinsults. These secondary effects largely determine the long-termfunctional outcome.

The immediate injury-induced response strongly influences the subsequentdegenerative response. Treatment that reduces or attenuates the injuryto the primary insults is therefore likely to generate optimal resultsby preventing or delaying the secondary degenerative processes.

It has now been discovered that neuroprotection is conferred uponretinal neurons by administration of a ryanodine antagonist, e.g.dantrolene, to the retina of a mammal within a period prior to, orfollowing an primary insult to the retinal neurons but prior to celldeath.

The terms noxious actions or noxious provocations are defined as anoccurrence which is harmful or destructive to a nerve cell. It is notlimited to events extrinsic to the mammal being treated but includesdisease states and pathological occurrences or events, such as, forexample, stroke or heart attack, that are harmful or destructive to thenerve cell via a chain of events. Non-limiting examples of noxiousactions include: compressive or mechanical effects or trauma or stressfactors, such as glutamate neurotoxicity, impaired blood flow to thenerves (ischemia) and with respect to the retina, glaucoma, diabeticretinopathy, retinitis pigmentosa and age-related macular degeneration.

Human Dosage and Administration

The methods of this invention are useful in treating any mammal,including humans.

According to this invention, mammals are treated with pharmaceuticallyeffective amount of a neuroprotective agent for a period of time and ata time such that noxious provocations do not kill or permanently damagethe nerve cells. Protective agents may be administered orally, topicallyto the eye or by any other appropriate means of delivery described belowor known in the art.

In accordance with this invention, pharmaceutically effective amounts ofa protective agent can be administered alone to treat neural injury orto prevent nerve cell death. Alternatively a protective agent may beadministered sequentially or concurrently with another drug. Forexample, it may be used with an antiglaucoma drug, such as abeta-blocker, an alpha₂ agonist, a muscarinic agent such as pilocarpine,a carbonic anhydrase inhibitor (CAI), or other intraocular pressure(TOP) lowering drugs. It may also be used with an anti-angiogenesis drugfor the treatment of ARMD and diabetic retinopathy. The most effectivemode of administration and dosage regimen of protective agent willdepend on the type of disease to be treated, the severity and course ofthat disease, previous therapy, the patient's health status, andresponse to the drug and the judgment of the treating physician.Generally, the neuroprotective agent should be administered in a dose toachieve a serum or intravitreal concentration of 0.01 nM to 5 M.Preferably the neuroprotective agent is administered prior to injury tothe nerve, but can be administered after injury has occurred withlessened effect.

Conventional modes of administration and standard dosage regimens ofneuroprotective agents can be used. Optimal dosages for coadministrationof a drug, e.g. an IOP-lowering drug, with a neuroprotective agent canbe determined using methods known in the art. Dosages of neuroprotectiveagents may be adjusted to the individual patient based on the dosage ofthe drug with which the agent is coadministered and the response of thepatient to the treatment regimen. The neuroprotective agent may beadministered to the patient at one time or over a series of treatments.

The agent may be administered locally, e.g. intravitreally byintrabulbar injection for ocular neuroprotection, or by intrathecal orepidural administration for spinal protection. Many of the agents of theinvention can be administered systemically, e.g., orally, orintravenously, or by intramuscular injection. Additionally, agents forprotection of the retina and optic nerve that are capable of passingthrough the cornea, and achieving sufficient concentration in thevitreous humor, may also be administered topically to the eye.

The composition used in these therapies may also be in a variety offorms. These include, for example, solid, semi-solid, and liquid dosageforms, such as tablets, pills, powders, preserved or non-preservedliquid solution or suspension, liposomes, suppositories, injectable andinfusible solutions. The compositions also preferably includeconventional pharmaceutically acceptable carriers which are known tothose of skill in the art.

The following non-limiting examples describe assays and measurementsused in 1) evaluating efficacy of neuroprotecting agents and 2)selecting ryanodine antagonists other than dantrolene.

Example 1:

Experimental Procedure for Measuring Neural Protection in Rabbit Model.

To evaluate in vivo neuroprotective effects of dantrolene (DTL) onNMDA-induced injury of RGCs an imaging method to count cell numbers atthe RGC layer in the isolated retinas was developed. Briefly, two weeksfollowing intravitreal injection of vehicle or various test agents, arabbit was euthanized and the treated eye was enucleated. A piece ofretina (8 mm in diameter) was cut immediately below the optic nervehead, flat-mounted in a plastic chamber filled with HEPES-buffered Amesmedium, and imaged at 25 fields in a 5×5 array with a 40× waterimmersion objective using an Olympus microscope (BX50WI) equipped withan epi-fluorescence unit. The images were taken with a HamamatsuC4742-95 digital camera and Image-Pro Plus software (V4.5). The totalnumber of neurons at the ganglion cell layer in these 25 fields wascounted. The same measurements were conducted in one control group(rabbits treated with vehicle) and 4 test groups treated with 1) NMDA,2) dantrolene+NMDA, 3) veratridine (Verat) a neurotoxin that damagesretinal ganglion cells with intracellular sodium overload, and 4)dantrolene+veratridine. The results from the 4 test groups arenormalized with respect to that of control.

The results are reported in FIG. 1. NMDA caused a loss of 53% of cellsat the RGC layer. Pretreatment with dantrolene significantly reducedNMDA-induced cell death to 35%. Dantrolene also reduced cell loss causedby veratridine from 56% to 50%.

Example 2:

Assay for Selecting Ryanodine Antagonists other than Dantrolene.

Assays for determining ryanodine antagonist may be conducted followingprocedures modified from that described by Laver et al., (J. Physiol.537:763-778, 2001). Briefly, purified ryanodine receptor-channelcomplexes are incorporated into planar phospholipid bilayers withresting calcium gradient similar to that in a normal neuron at rest (100nM cytoplasmic and 1 mM luminal). The level of channel activation can bedetermined in the presence of various ligands that activate ryanodinereceptors. Effective antagonistic action of the compounds to be selectedcan be determined by a reduction of agonist-induced activation of thechannel. The specificity of the antagonists can be determined bycommercially available standard screens, such as NovaScreens.

While this invention has been described with respect to various specificexamples and embodiments, it is to be understood that the invention isnot limited thereby and should only be construed by interpretation ofthe scope of the appended claims.

1-16. (canceled)
 17. A method for treating retinal ganglion cell loss ina patient comprising administering dantrolene to said patient.
 18. Themethod according to claim 17, wherein said administering comprises oraladministration.
 19. The method according to claim 17 wherein saidadministering comprises intravitreal injection.
 20. The method accordingto claim 17 wherein said administering comprises topical administrationof an ophthalmic solution of the dantrolene to the eye.
 21. The methodaccording to claim 17, wherein the dantrolene is in the form of anaqueous solution, a suspension, a gel, or a jelly.